Heparan sulfate proteoglycans (HSPGs) are macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues (1-3). Heparan sulfate (HS) binds to and assembles ECM proteins and plays important roles in the structural integrity of the ECM and in cell-cell and cell-ECM interactions. HS chains sequester a multitude of proteins and bioactive molecules and thereby function in the control of a large number of normal and pathological processes (1-4). Apart from sequestration of bioactive molecules, HSPGs have a coreceptor role in which the proteoglycan, in concert with the other cell surface molecule, comprises a functional receptor complex that binds the ligand and mediates its action (3-5).
Enzymatic degradation of HS by heparanase, a mammalian endoglucuronidase, affects the integrity and functional state of tissues and is involved in fundamental biological phenomena, ranging from pregnancy, morphogenesis and development to inflammation, angiogenesis and cancer metastasis (6-10). Heparanase elicits an indirect angiogenic response by releasing HS-bound angiogenic growth factors (e.g., basic fibroblast growth factor—bFGF and vascular endothelial growth factor—VEGF) from the ECM and by generating HS fragments that potentiate bFGF receptor binding, dimerization and signaling (5, 8).
By degradating HS of cell surface and ECM, heparanase facilitates locomotion of inflammatory and tumor cells, release growth factors bound to the ECM, and induce new blood vessels formation (angiogenesis). Heparanase expression in tumor cells is correlated with worse prognosis, and its expression in experimental tumor models resulted in increased tumor growth and metastasis formation. Moreover, elevated levels of heparanase have been detected in sera of animals and human cancer patients bearing metastatic tumors, and in the urine of some patients with aggressive metastatic disease. Regulation of heparanase activity in normal tissues is poorly understood.
Despite earlier reports on existence of several distinct mammalian HS-degrading endoglycosidases (heparanases), the cloning of the same single gene (SEQ ID NO: 41) by several groups (6, 7, 11, 12) suggests that mammalian cells express primarily a single dominant functional heparanase enzyme. Since the cloning of human heparanase, no splice variants were described.
Human heparanase is synthesized as a latent 65-kDa precursor whose processing involves proteolytic cleavage and formation of an active enzyme composed of two 50-kDa and 8-kDa subunits (13-15).
Heparanase exhibits endoglycosidase activity at acidic pH (5-6.7), which exists in nonvascularized core of tumor masses. Heparanase mRNA is increased in human breast, colon, lung, prostate, ovary and pancreas tumors compared with the corresponding normal tissues. In human normal tissues, heparanase mRNA expression is limited to the placenta and lymphoid organs.
Because heparanase promotes angiogenesis and cancer progression, the present inventors found of interest to investigate the evolution of this unique enzyme in a wild mammal that was exposed to underground hypoxic stress throughout the family Spalacidae evolutionary history (16).
The subterranean blind mole rat of the genus Spalax in Israel, belongs to the superspecies Spalax ehrenbergi, consisting of at least 12 allospecies in the Near East. The four Israeli species have been the subject of intensive and extensive interdisciplinary evolutionary studies (16, 17). They represent four species with different diploid chromosome number (2n) associated with four climatic regimes in Israel. These include: Spalax galili (2n=52), which lives in the humid-cool upper Galilee mountains; S. golani (2n=54), which lives in the semidry, cool Golan heights; S. carmeli (2n=58), which ranges in humid-warm central Israel; and S. judaei (2n=60), which lives in the dry and warm Samaria, Judea, and the northern Negev (16-18). Spalax lives all its life, averaging three years, in sealed underground tunnels (19), evolving a unique adaptive complex to cope with hypoxia and hypercapnia (20, 17).
Among the strategies used by Spalax to tolerate hypoxia are: higher myocardial maximal oxygen consumption (21), structural adaptations in tissues that result in a decreased diffusion distance of oxygen to the mitochondria (22), increase in the lung diffusion capacity (22), specific differences in myoglobin which augment oxygen delivery at low oxygen tensions (23), and increased density of blood vessels, correlated with a unique VEGF expression pattern (19, 24, 25). Hemoglobin and hematocrit are higher in the northern species which survive more hypoxia than the southern ones (17).
The present inventors have recently cloned and elucidated the expression of p53 (26, 27) and VEGF (24, 25) in Spalax. p53 gene in healthy Spalax individuals possesses two amino acid substitutions in its DNA binding domain, identical to mutations found in human tumors. These adaptive substitutions endow Spalax p53 with several-fold higher activation of cell arrest and DNA repair genes compared to human p53, and they also favor activation of DNA repair genes over apoptotic genes. Expression of VEGF was constitutively high in Spalax muscles, regardless of the oxygen levels, similar to its expression in highly metastatic tumor cells (28) and unlike its levels in rat muscle (25).